New tools to probe iron and manganese homeostasis in bacterial and eukaryotic cells

项目摘要 除了极少数例外，催化自然界最具挑战性和最重要反应的酶都需要 活泼的金属离子。在从细菌到人类的生物体中，最重要的金属是铁 和锰：几乎所有的生物都需要铁来生长，少数不需要铁的生物依赖于 相反，它是锰。细胞仔细地调节它们所需的金属离子的浓度 摄取蛋白、出口蛋白、运输蛋白以及基于蛋白质和RNA的调控的复杂网络 系统。所有这些系统的正确运行对于避免潜在的有害化学物质是至关重要的 由于加入了不正确的金属离子而导致的“游离”金属离子以及酶的失活。作为一名 结果，对细胞内游离铁和锰浓度的管理不善与 人类疾病的数量，特别是神经退行性变。另一方面，寄主生物体的能力 (人类)破坏入侵病原体的金属管理与降低其毒力有关 这种病原体的。因此，对铁和锰的作用机理有了更详细的了解 细菌和人类体内的动态平衡有可能导致治疗这些疾病的新方法。一个 金属选择荧光的设计和应用是研究金属动态平衡的重要途径 传感器--由小分子、蛋白质或核酸组成--能够报告 细胞内金属离子的浓度、定位和动力学。金属的荧光传感器，如 钙和锌彻底改变了我们对这些离子生物学的理解。不幸的是，很少有工具 存在于研究细胞内的铁和锰，反映了与选择性相关的内在挑战 检测生物系统中倾向于弱结合的分析物。这项研究提案概述了一个 综合计划，开发铁和锰的选择性荧光传感器，使用自然的 选择性金属识别平台，以探索细菌和细菌中金属调控的新机制 在真核细胞中。该提案使用了铁和铁识别金属的详细生化分析 结合锰的蛋白质和核酸，以合理设计新的传感器。这些传感器就是 部署以深入了解该领域的悬而未决的问题，如 病原菌中铁超载以及细胞内锰转运系统的特征 在酵母和人类细胞中。这些答案不仅将增加我们对基本机制的理解 在生物系统中的选择性金属识别，但也潜在地发现了治疗的新途径 干预。

Project Summary With few exceptions, the enzymes that catalyze Nature’s most challenging and important reactions all require metal ions for activity. Among the most important of these metals in organisms from bacteria to humans are iron and manganese: nearly all organisms require iron for growth, and the few that do not require iron depend on manganese instead. Cells carefully regulate the concentrations of the metal ions that they require through a complex network of uptake proteins, export proteins, trafficking proteins, and protein- and RNA-based regulatory systems. The proper function of all of these systems are essential to avoid potentially deleterious chemistry of the “free” metal ions as well as inactivation of enzymes resulting from incorporation of incorrect metal ions. As a result, mismanagement of the cellular free concentrations of iron and manganese has been connected with a number of human diseases, in particular neurodegeneration. On the other hand, the ability of a host organism (a human being) to disrupt metal management in an invading pathogen correlates with reduction of the virulence of that pathogen. Therefore, a more detailed understanding of the mechanisms of iron and manganese homeostasis in bacteria and humans has the potential to lead to new approaches to treat these diseases. An important approach to study metal homeostasis involves design and application of metal-selective fluorescent sensors – consisting of either small molecules, proteins, or nucleic acids – which are able to report on concentration, localization, and dynamics of the metal ions within cells. Fluorescent sensors for metals such as calcium and zinc have revolutionized our understanding of the biology of these ions. Unfortunately, few tools exist to study iron and manganese within cells, a reflection of the inherent challenges associated with selectively detecting an analyte that tends to bind weakly in biological systems. This research proposal outlines a comprehensive program to develop selective fluorescent sensors for iron and manganese, using Nature’s platforms for selective metal recognition, in order to probe novel mechanisms of metal regulation in bacteria and in eukaryotic cells. The proposal uses detailed biochemical analysis of metal recognition by iron- and manganese-binding proteins and nucleic acids to rationally design new sensors. These sensors are then deployed to provide insight into outstanding questions in the field, such as the mechanism and consequences of iron overload in pathogenic bacteria, as well as characterization of intracellular manganese trafficking systems in yeast and human cells. These answers will not only increase our understanding of fundamental mechanisms of selective metal recognition in biological systems but also potentially uncover new avenues for therapeutic intervention.